Abstract

In the present study, we investigated a new member of the ABC transporter superfamily of Arabidopsis thaliana, AtMRP5. AtMRP5 encodes a 167 kDa protein and exhibits low glutathione conjugate and glucuronide conjugate transport activity. Promotor- beta-glucuronidase fusion constructs showed that AtMRP5 is expressed mainly in the vascular bundle and in the epidermis, especially guard cells. Using reverse genetics, we identified a plant with a T-DNA insertion in AtMRP5 (mrp5-1). mrp5-1 exhibited decreased root growth and increased lateral root formation. Auxin levels in the roots of mrp5-1 plants were increased. This observation may indicate that AtMRP5 works as an auxin conjugate transporter or that mutant plants are affected in ion uptake, which may lead to changes in auxin concentrations. Experiments on epidermal strips showed that in contrast to wild type, the sulfonylurea glibenclamide had no effect on stomatal opening in mrp5-1 plants. This result strongly suggests that AtMRP5 may also function as an ion channel regulator.

Fig. 1. Sequence and genomic structure of AtMRP5. (A) The predicted AtMRP5 protein sequence. Lower case letters of the gene sequence indicate the 5′-non-translated sequence. Putative transmembrane-spanning domains identified using the TMpred program () are underlined; the Walker motifs A and B, as well as motif C, are represented as small grey boxes. The methionine, incorrectly annotated in gene F20D22.11 (DDBJ/EMBL/GenBank accession No. AC002411) as being the first amino acid, is boxed. (B) Genomic organization of the AtMRP5 gene as deduced from the cDNA and a corresponding genomic sequence located on BAC F20D22. The promoter (arrow), as well as the 5′- and 3′-UTRs are shown as black boxes; exons are presented as dark grey, introns as light grey boxes. Exon and intron sizes are given in bold and standard letters, respectively. The insertion site and the orientation of the T-DNA in the mrp5-1 mutant is indicated.

Fig. 3. Histochemical localization of GUS activity. (A) A seedling at 7 days after germination (dag) showing GUS expression in cotyledons and vascular tissue in the tip of primary leaves. (B) The leaf of an Arabidopsis plant 21 dag exhibiting GUS expression in lower and higher order veins. (C) Dark-field observation of a cross-section of an adult leaf with GUS expression in vascular tissue, epidermal cells and weakly in mesophyll cells. (D) The abaxial epidermis of an adult leaf exhibits strong GUS staining in guard cells. (E) Flower petals showing GUS expression in guard cells. (F) GUS staining in pollen sacs is present along the central vascular strand of the filament and in connecting tissue. (G) The root of a seedling at 11 dag. GUS expression is present in the central cylinder but not in root tips. (H) GUS staining at the pod attachment site.

Fig. 4. DNA blot analysis of six singular F2 plants of the mrp5-1/Ws-2 crossing exhibiting mrp5-1/mrp5-1 (lanes 1 and 2 in B and C), mrp5-1/Ws-2 (lanes 3 and 4) and Ws-2/Ws-2 (lanes 5 and 6) genotypes (A–C) and RT–PCR analysis of mrp5-1/mrp5-1 plants (D). (A) Schematic view of the 5.6 kb genomic sequence of AtMRP5 and of the 17 kb T-DNA construct 3850:1003 () inserted in mrp5-1 (triangle) with predicted restriction sites of enzymes EcoRI (E), HindIII (H) and BamHI (B) (numbers indicate relative positions in kb). The position of the gene-specific probe and a probe specific for the T-DNA left border are indicated as boxes denoted A and T, respectively. (B) DNA blot analysis of genomic DNA digested with EcoRI, HindIII and BamHI probed with the gene-specific probe A. (C) The same as (B) using the T-DNA probe T. The arrow highlights the 15 kb band visible after restriction with BamHI. (D) RT–PCR analysis of AtMRP5 and S16 expression in Ws-2/Ws-2 and mrp5-1/mrp5-1 plants.

Fig. 6. Stomata of mrp5-1 plants are insensitive towards the sulfonylurea glibenclamide. (A) The change in stomatal aperture was measured as the difference between aperture values in the presence and absence of 8 µM glibenclamide. Each column represents the mean of five independent experiments (±SEM) each conducted on five plants. The aperture of 60 stomata was determined per experiment. Individual stomata exposed for 3 h are illustrated in the respective columns. (B) A representative experiment showing that application of glibenclamide for 3 h in the dark produces a dose-dependent increase in stomatal aperture in the wild-type plant (open squares) but not in the mrp5-1 plants (solid circles). Half-maximal opening of stomata is at 0.8 µM glibenclamide.